Extensive evidence suggests that oxidative modification of biological molecules, such as lipids and proteins, plays a significant role in many human diseases and aging. The mechanisms of protein oxidation, however, are not well understood. The goal of this research was to study the molecular basis of in vitro protein oxidation induced by a free-radical initiator. Purified Escherichia coli glutamine synthetase was subjected to oxidation by incubation with azo-bis amidinopropane HCL (AAPH) at 37 degrees C. The enzyme activity and hydroperoxide formation were determined. Oxidized proteins were identified using SDS-polyacrylamide gel electrophoresis and analyzed for amino acid composition using the O-phthalaldehyde method with HPLC on a C18 column. AAPH is a water soluble azo-initiator, which decomposes to generate free radicals at a known and constant rate. At physiological pH, AAPH inactivated glutamine synthetase and produced hydroperoxide as a function of time. As revealed by SDS-PAGE, co-incubation of glutamine synthetase with AAPH led to protein aggregation. This finding was consistent with the results of gel filtration where oxidized glutamine synthetase passed through a Sepharose CL-6B column earlier than control glutamine synthetase. Oxidative damage to glutamine synthetase also resulted in the loss of some histidine, methionine, and tyrosine residues. The size and other characteristics of oxidized glutamine synthetase are currently being investigated.